Upregulation of human GD3 synthase (hST8Sia I) gene expression during serum starvation-induced osteoblastic differentiation of MG-63 cells

In this study, we have firstly elucidated that serum starvation augmented the levels of human GD3 synthase (hST8Sia I) gene and ganglioside GD3 expression as well as bone morphogenic protein-2 and osteocalcin expression during MG-63 cell differentiation using RT-PCR, qPCR, Western blot and immunofluorescence microscopy. To evaluate upregulation of hST8Sia I gene during MG-63 cell differentiation by serum starvation, promoter area of the hST8Sia I gene was functionally analyzed. Promoter analysis using luciferase reporter assay system harboring various constructs of the hST8Sia I gene proved that the cis-acting region at -1146/-646, which includes binding sites of the known transcription factors AP-1, CREB, c-Ets-1 and NF-κB, displays the highest level of promoter activity in response to serum starvation in MG-63 cells. The -731/-722 region, which contains the NF-κB binding site, was proved to be essential for expression of the hST8Sia I gene by serum starvation in MG-63 cells by site-directed mutagenesis, NF-κB inhibition, and chromatin immunoprecipitation (ChIP) assay. Knockdown of hST8Sia I using shRNA suggested that expressions of hST8Sia I and GD3 have no apparent effect on differentiation of MG-63 cells. Moreover, the transcriptional activation of hST8Sia I gene by serum starvation was strongly hindered by SB203580, a p38MAPK inhibitor in MG-63 cells. From these results, it has been suggested that transcription activity of hST8Sia I gene by serum starvation in human osteosarcoma MG-63 cells is regulated by p38MAPK/NF-κB signaling pathway.

Among human sialyltransferases, seven enzymes including hB4Gal T I, hB3GalT IV, hST3Gal II, hST3Gal III, hST3Gal V, hST8Sia I and hST8Sia V are involved in biosynthetic pathway of the gangliosides [11,12].Our previous studies have shown that serum starvation also triggers cell differentiation of human MG-63 osteoblastic cells [9,10].Moreover, we found that during MG-63 cell differentiation, serum starvation increased gene expressions of the human β-galactoside α2,6-sialyltransferase (hST6Gal I) [10], as well as the human GM3 synthase (hST3Gal V), also called lactosylceramide α2,3-sialyltransferase, which catalyzes biosynthesis of ganglioside GM3 [9].Interestingly, during our studies on serum starvationinduced expression of human sialyltransferases, expression of hST8Sia I gene was particularly increased, being the highest level.Based on these findings, we continued to investigate the gene expression of ganglioside-specific sialyltransferases during osteoblast differentiation.
In the present study, the expression of hST8Sia I, known as Sia:GM3 α2,8-sialyltransferase, which catalyzes the formation of disialoganglioside GD3 from monosialoganglioside GM3, as a key biosynthetic enzyme of the b-and c-series gangliosides [11,12], specifically increases during the differentiation of MG-63 cells by serum starvation.Furthermore, the distinct elevation of hST8Sia I protein level was observed in serum starvation-induced MG-63 cells.In parallel with the hST8Sia I gene, we examined whether serum starvation alters in the expression of differentiation marker molecules of BMP-2 and osteocalcin at the mRNA levels in MG-63 cells.In conclusion, we have here elucidated transcriptional upregulation of hST8Sia I gene responsible for the increment of its gene expression induced by serum starvation.

Cell cultures
Cell culture of human MG-63 and U2OS osteoblastic cells was conducted as described previously [9,10] using Dulbecco's modified Eagle's medium (DMEM; WelGENE Co., Daegu, Korea) supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 10% (v/v) fetal bovine serum (FBS) (Gibco BRL, Life Technologies; Grand Island, NY, USA).To induce cell differentiation and the increased expression of hST8Sia I gene, the cell was placed in FBSfree medium for different time periods.

Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR)
Isolation of total RNA from cultured cells, first-strand cDNA synthesis, and PCR amplification were performed as previously described [9,10].Six kinds of ganglioside-specific human sialyltransferase genes were amplified by PCR condition with specific primers shown in S1 Table .Time-dependent RT-PCR of specific hST3Gal V, bone morphogenic protein-2 (BMP-2), osteocalcin and β-actin was conducted with primers shown in Table 1.PCR products were analyzed by 1% agarose gel electrophoresis.Real-time qRT-PCR was performed as previously described [9,10] using a CFX96 TM Real-Time system with SYBR Premix (Bio-Rad) and the specific primers as shown in Table 1.Target gene expression was normalized to β-actin mRNA expression.

Immunofluorescence staining
Immunofluorescence staining was conducted as previously described [18].MG-63 cells grown for 24 h on sterile coverslips containing medium with or without 10% FBS were fixed with 4% paraformaldehyde for 10 min at 37˚C, washed three times with PBS, and blocked with 5% BSA for 1 h at 37˚C.After incubation for overnight at 4˚C with the anti-GD3 monoclonal antibody (mAb) (mouse IgM, Kappa-chain, clone, GMR19; Seigakagu, Tokyo, Japan), cells were reacted with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG/M/A mixture (Vector labs, F1-1000) used as the secondary antibody for 1 h at 37˚C.The nucleus was stained with DAPI for 10 min at room temperature.Fluorescence images of cells were obtained by using LSM 700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

Western blot analysis
Western blot analysis was conducted as described previously [9].The cell lysates obtained by lysis in RIPA buffer were separated on SDS-polyacrylamide gels and transferred to PVDF membrane.Membrane was incubated with sequentially with hST8Sia I-specific antibody (#sc-46982, Santa Cruz, CA) and horseradish peroxidase (HRP)-conjugated secondary antibody (Enzo Life Science, Farmingdale, NY).GAPDH (#sc-20357, Santa Cruz, CA) was used as an internal control.Blots were detected using the ECL chemiluminescence system (GE Healthcare, Piscataway, NJ, USA).

Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed using a ChIP assay kit (Millipore, USA) according to the manufacturer's instruction.As described previously [16][17][18], cells were cross linked in 1% formaldehyde at 37˚C for 10 min to cross-link DNAs and protein, and then sonicated to shear genomic DNAs to average size of 200-1000 bp.Immunoprecipitation was carried out using 4 μg of NF-κB antibody (Santa Cruz Biotechnology) and IgG antibody (Sigma) as negative control.The purified ChIP DNA or input DNA was used for PCR analysis using primers flanking NF-κB binding site (-731/-722) on the hST8Sia I promoter (Table 1).

Knockdown of hST8Sia I
Lentiviral plasmid pLKO.1 containing shRNA targeting hST8Sia I (TRCN0000036045) or empty vector pLKO.1 (SHC001) were purchased from Sigma-Aldrich.Lentivirus productions and lentiviral infections were performed according to the supplier's protocol.After puromycin selection to generate stable cell lines with empty vector shRNA as a control and hST8Sia I-specific shRNA, cells were cultivated for 24 h in FBS-free medium and the knock-down efficacy of hST8Sia I shRNA was assessed by real-time qRT-PCR and Western blot analysis.

Serum starvation influences the gene expression of ganglioside-specific human sialyltransferases
It is well known that seven kinds of human sialyltransferases (hB4Gal T I, hB3GalT IV, hST3Gal II, hST3Gal III, hST3Gal V, hST8Sia I, hST8Sia V) are involved in ganglioside biosynthesis [11,12].Because we showed increased gene expression of hST3Gal V during MG-63 cell differentiation by serum starvation in a previous study [9], the effects of serum starvation on the gene expression of six human sialyltransferases, excluding hST3Gal V, were investigated by RT-PCR.Serum starvation increased the mRNA levels of the five types of human sialyltransferases, except for hST8Sia V.Among them, the expression level of hST8Sia I was the highest, followed by hST3Gal II, but hB4GalT I, hB3GalT IV, and hST3Gal III showed low levels (S1 Fig) .Based on this result, the mechanism of upregulation of hST8Sia I expression by serum starvation was further investigated in this study.

Gene expression of hST8Sia I by serum starvation in human osteosarcoma MG-63 cells
In previous studies, we revealed that the expression of BMP-2 and osteocalcin, well-known markers of osteoblastic differentiation, increased in a time-dependent manner by serum starvation in MG-63 cells [9,10].In this study, as shown in

Ganglioside GD3 expression by serum starvation in human osteosarcoma MG-63 cells
To investigate whether the ganglioside GD3 level produced by hST8Sia I was increased by serum starvation in MG-63 cells, we analyzed ganglioside GD3 expression at the cellular level using an anti-GD3 monoclonal antibody (mAb) and a secondary antibody (FITC-conjugated goat-anti-mouse IgM) by immunofluorescence confocal microscopy.As a result, the increased GD3 expression level was observed in cells cultivated for 24 h in an FBS-free medium, but not in a 10% FBS-containing medium (Fig 2A).This result suggests that the increased gene expression of hST8Sia I induced by serum starvation contributes to an elevation of ganglioside GD3 expression at the cellular level.

Comparison of serum starvation-induced transcriptional activity of hST8Sia I gene promoter in MG-63 cells
To elucidate whether the mRNA levels of hST8Sia I were markedly enhanced by its promoter activity induced by serum starvation in MG-63 cells (Fig

Effect of ganglioside GD3 on osteoblast differentiation
To investigate whether ganglioside GD3 synthesized by hST8Sia I exerts an influence on osteoblast differentiation, hST8Sia I was knocked down shRNA and then the changes in the mRNA levels of hST8Sia I and osteoblast differentiation markers, BMP-2, alkaline phosphatase(ALP), and osteocalcin were checked by real-time qRT-PCR analysis.hST8Sia I mRNA expression levels were remarkably reduced by 5.7-fold in hST8Sia I knockdown cells compared to control cells (Fig 6A ), whereas in hST8Sia I knockdown cells, the mRNA levels of BMP-2, ALP, and osteocalcin were 1.6-, 2.4-and 4.8-folds higher than those in control cells.In addition, protein levels of hST8Sia I and ganglioside GD3 were distinctly decreased in hST8Sia I knockdown cells compared to control cells (Fig 6B and 6C).Collectively, these results suggest that expression of hST8Sia I and ganglioside GD3 have no apparent effect on the differentiation of MG-63 cells.

Discussion
Although in human sialyltransferases, gene expression directly associated with ganglioside biosynthesis has been widely reported in embryonic development, inflammation, degeneration, cancer progression, and metastasis [19][20][21][22], little is known about their gene expression in relation to osteoblast differentiation.We have previously demonstrated that serum starvation induces cell cycle arrest at the G1 stage preceding cell differentiation of MG-63 cells, but has no significant effect on cell proliferation, about 73% viability even after serum starvation for 48 h [9].In this study, we analyzed the expression of six human sialyltransferases (hB4Gal T I, hB3GalT IV, hST3Gal II, hST3Gal III, hST8Sia I, and hST8Sia V) involved in ganglioside biosynthesis during serum starvation.Our RT-PCR results showed that the level of hST8Sia I expression was highest in serum starvation-induced MG-83 cells.In our previous studies, we reported for the first time serum starvation-induced osteoblastic differentiation of MG-63 cells and demonstrated that serum starvation significantly elevated the mRNA levels of osteoblast differentiation markers, such as BMP and osteocalcin, along with high expression of hST6Gal I and hST3Gal V [9,10].In line with these observations, we also found that the transcript levels of BMP and osteocalcin increased in a time-dependent manner, along with high mRNA levels of hST8Sia I, as demonstrated by RT-PCR and qPCR, suggesting osteoblastic differentiation by serum starvation in MG-63 cells.
In the present study, the effect of serum starvation on osteoblast differentiation in vitro was investigated using MG-63 cells.We have demonstrated that expression of hST8Sia I at both the mRNA and protein levels was significantly augmented by serum starvation in a timedependent manner, as confirmed by western blot, RT-PCR, and qPCR analysis, indicating upregulation of hST8Sia I by serum starvation at both transcriptional and translational levels in MG-63 cells.Interestingly, gene expression of hST8Sia I by serum starvation is clearly similar to that of hST6Gal I [10], in that mRNA levels of hST8Sia I begin to increase after 6 h and are maximal at 48 h.Moreover, we also demonstrated that elevated hST8Sia I expression correlates with increased levels of ganglioside GD3 observed after serum starvation for 24 h in MG-63 cells, as confirmed by immunostaining using GD3 mAb.
In the present study, we clarified that the nt -1146 to -646 promoter region of hST8Sia I functions as a serum starvation-responsive core promoter by a luciferase assay using deletion constructs.This region also plays the role of a core promoter crucial for the transcriptional upregulation of hST8Sia I in Fas-induced Jurkat T cells [13], human melanoma SK-MEL-2 cells [14], valproic acid-induced SK-N-BE(2)-C human neuroblastoma cells [15], cordycepininduced SK-N-BE(2)-C human neuroblastoma cells [16], and curcumin-induced A549 human lung cancer cells [17].
Consistently, we also clarified that the NF-κB binding site (-731/-722) in the-1146/-646 region is critical for the transcriptional upregulation of hST8Sia I in serum starvation-induced MG-63 cells, as evidenced by site-specific mutagenic analysis, NF-κB-specific inhibitor (PDTC) treatment, and in vivo ChIP assay.Of particular interest is our finding that this NF-κB binding site (-731/-722) essentially functions as a major regulator of the transcriptional upregulation of hST8Sia I by various extracellular stimuli in different cancer cell types [13][14][15][16].In contrast, we previously confirmed that this NF-κB binding site (-731/-722) mediates transcriptional repression of hST8Sia I by triptolide in SK-MEL-2 human melanoma cells [18].In addition, Bobowski et al. also proved that hST8Sia I transcription is suppressed through the inhibition of NF-κB activation by estradiol in human breast cancer cells expressing the estrogen receptor α (ERα) [23].
In previous studies, we have reported that transcriptional upregulation of hST8Sia I is caused by Fas signaling in Jurkat T cells [13] and AMPK signaling in A549 human lung cancer cells [17].In contrast, in the present study, we revealed that the transcriptional upregulation of hST8Sia I by serum starvation in MG-63 cells is mediated by the p38 MAPK signaling pathway, as featured by the p38MAPK inhibitor SB203580.Previous studies showed that the p38MAPK signal pathway is involved in osteoblast differentiation [24][25][26][27].Moreover, activation of the p38MAPK pathway triggered NF-κB activation during osteoblastic cell differentiation [28,29].Thus, it may be assumed that the upregulation of hST8Sia I transcription by serum starvation in MG-63 cells is mediated by the p38MAPK/NF-κB signaling pathway.
Some studies have reported that ganglioside GD3 an important role in cell differentiation [29][30][31][32].For example, it has been reported that GD3 expression by transfection of mouse ST8Sia I cDNA into a murine neuroblastoma cell line, Neuro2a, provoked cell differentiation with neurite sprouting [28,29].Our group previously demonstrated that GD3 expression by transfection of hST8Sia I cDNA into K562 leukemia cells caused erythroid differentiation [30].A significant elevation of GD3 expression with GD1a was observed during neural differentiation of human dental pulp stem cells (hDPSCs) [31].Although it has been reported that during osteoblastic differentiation of human mesenchymal stem cells (hMSCs), suppression of ganglioside GD1a expression by shRNA-mediated knockdown of hST3Gal II catalyzing GD1a synthesis caused a remarkable reduction in ALP activity, which suggests the hindrance of osteoblastic differentiation by suppression of GD1a [32], the effect of suppression of GD3 expression on osteoblastic differentiation has yet to be reported.In this study, we investigated for the first time whether suppression of GD3 expression influences osteoblastic differentiation in human osteosarcoma MG-63 cells.Suppression of GD3 expression by knockdown of hST8Sia I did not cause any reduction in gene expression levels of BMP, ALP, and osteocalcin known as osteoblast differentiation markers.This result suggests that unlike the effect of GD1a suppression by hST3Gal II knockdown, suppression of GD3 expression by hST8Sia I knockdown had no apparent influence on osteoblastic differentiation in MG-63 cells.

Conclusions
Our study proved for the first time that the levels of hST8Sia I and ganglioside GD3 are elevated during human osteoblastic MG-63 cell differentiation by serum starvation.Furthermore, we elucidated the transcriptional upregulation of hST8Sia I during serum-starvation-triggered MG-63 cell differentiation by promoter analysis using a luciferase reporter assay system.We verified that the -731/-722 sequence, including the NF-κB binding site, is essential for hST8Sia I expression induced by serum starvation in MG-63 human osteosarcoma cells by site-directed mutagenesis, NF-κB inhibition, and ChIP assays.Taken together, our results revealed that the transcriptional upregulation of hST8Sia I in serum-starvation-induced MG-63 cells is mediated by the p38MAPK/NF-κB signal pathway.The present results also support the conclusion that isolated osteoblasts are sensitive to serum starvation and that serum starvation increases osteoblast activity in that expression of differentiation-related genes in response to serum starvation is increased in cultured MG-63 cells.In addition, our results suggest that the expression of hST8Sia I and ganglioside GD3 has no apparent effect on the differentiation of MG-63 cells by shRNA-mediated hST8Sia I knockdown.

Fig 1 ,
we verified that the expression levels of BMP-2 and osteocalcin were augmented by serum starvation in MG-63 cells in a time-dependent manner.In parallel, the time-dependent elevation of hST8Sia I expression by serum starvation was confirmed by RT-PCR and qPCR.These results indicated that the level of hST8Sia I expression was upregulated during MG-63 cell differentiation triggered by serum starvation.We observed similar results in the human osteoblastic cell line U2OS (S2 Fig).Furthermore, a distinct elevation of hST8Sia I protein levels in serum starvation-induced MG-63 cells compared with uninduced cells was observed by western blot analysis using an hST8Sia Irecognizing antibody (Fig 2B).

Fig 1 .
Fig 1.Effect of SS on expression levels of hST8Sia I and osteoblastic markers.Total RNA from MG-63 cells was isolated after incubation in serumfree medium for the indicated time periods and mRNA transcripts of hST8Sia I and osteoblastic markers (BMP-2 and osteocalcin) were detected by RT-PCR (A) and quantitative real-time PCR analysis (B-D).As an internal control, parallel reactions were performed to measure levels of the housekeeping gene β-actin.The transcript copy numbers of hST8Sia I and osteoblastic markers were normalized to the β-actin transcript copy number for each sample.Experiments were repeated three times to check reproducibility of results.*P < 0.05; **P < 0.01.***P < 0.001 (compared with control).https://doi.org/10.1371/journal.pone.0293321.g001

Fig 2 .
Fig 2. Confocal analysis of ganglioside GD3 expression and Western blot analysis of hST8Sia I in SS-induced MG-63 cells.(A) After incubation for 24 h in standard medium containing 10% FBS or 0% FBS, cells were immunostained with anti-GD3 antibodies (FITC; green).Nuclei were stained with DAPI (blue), and analyzed by confocal microscopy (100 ×).(B) Equal amounts of cell lysates (20 μg) were resolved in SDS-polyacrylamide gels and then transferred to PVDF membrane.Membrane was incubated with sequentially with hST8Sia I-specific antibody and horseradish peroxidase (HRP)-conjugated secondary antibody.GAPDH was used as an internal control.The bar graphs represent the intensities of the band obtained by densitometry.The values represent the means ± SEM of three independent experiments with triplicate measurements.*P < 0.05.https://doi.org/10.1371/journal.pone.0293321.g002

Fig 3 .
Fig 3. Analysis of hST8Sia I promoter activity in SS-induced MG-63 cells.The schematic diagrams represent DNA constructs (A) containing various lengths of the wild-type hST8Sia I promoter, or promoter construct (B) with mutant sequences in the 5'-flanking region, upstream of a luciferase reporter gene; the transcription start site is designated +1.The pGL3-basic construct, which did not contain a promoter or an enhancer, was used as a negative control.Each construct was transfected into MG-63 cells, with pRL-TK co-transfected as an internal control.The transfected cells were incubated in the presence (open bar) or absence (solid bar) of 10% FBS for 24 h.Relative firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay System, and all firefly activity was normalized to the Renilla luciferase activity derived from pRL-TK.The values represent the means ± SEM of three independent experiments with triplicate measurements.*P < 0.05; **P < 0.01.https://doi.org/10.1371/journal.pone.0293321.g003

Table 1 . Primer sequences used for RT-PCR, qPCR and ChIP in this study.
1), luciferase reporter plasmids (pGL3-1146/-646 to pGL3-2646/-646) were transfected into MG-63 cells and further incubated for 24 h in medium with or without 10% FBS and analyzed by luciferase assays.As shown in Fig3A, the four plasmids tested showed significantly increased luciferase activity in cells incubated in an FBS-free medium compared to those incubated in a medium with 10% FBS.Moreover, the luciferase activity obtained with the pGL3-1146/-646 construct revealed the highest increase (approximately 2.3-fold) compared to the other constructs.These results indicate that the nt -1146 to -646 region plays a crucial role in the serum starvation-responsive core promoter of hST8Sia I in MG-63 cells.